The endoplasmic reticulum (ER) stress response is frequently hyperactivated in cancer due to the accumulation of unfolded proteins, hypoxic conditions, calcium imbalance, and other stimuli associated with tumorigenesis. Inositol-requiring enzyme 1 (IRE-1) is a kinase/RNase that governs one of the three arms of the ER stress response through its regulation of the transcription factor XBP-1s. IRE-1 controls the expression of XBP-1s through its specific mRNA splicing activity, causing a frame-shift in translation. The resulting 54-kDA XBP-1s protein translocates into the nucleus and regulates ER stress response genes, contributing to normal B cell function and differentiation. Since gene copy number amplifications and aberrant protein expression are hallmarks of cancer, IRE-1 serves a critical cytoprotective function that allows tumor cells to evade stress- induced apoptosis. Building on our recent genetic and pharmacological validation of the IRE-1/XBP-1 pathway as a therapeutic target in CLL, we initiated an SAR campaign around novel tricyclic chromenones that form a stable and specific covalent complex with lysine 907 in the RNase domain of IRE-1. C-B06 (and its prodrug derivative, B-I09) potently inhibits XBP-1 mRNA splicing and suppresses XBP-1s expression in intact cells. Moreover, C-B06 and B-I09 are remarkably selective IRE-1 RNase antagonists that inhibit CLL cell growth in an XBP-1-dependant manner and reduce tumor burden in a transgenic mouse model of chronic lymphocytic leukemia (CLL) without imposing toxicity. Although a limited number of non-electrophilic allosteric inhibitors of IRE-1 RNase activity have also been reported, they have thus far not shown the ability to inhibit tumor cell growth in vitro on in vivo. This raises the possibiity that covalent inhibition of the IRE-1 RNase domain is required for anticancer efficacy. Here, we propose to use E-TCL1 mouse CLL cells, human CLL cell lines, and freshly isolated patient samples to guide the development of covalent IRE-1 RNase inhibitors as novel antileukemic agents. Moreover, we will assess the importance of specific covalent IRE-1 inhibition by comparing the activities of IRE-1 inhibitors with distinct mechanisms-of-action. In Aim 1, we will use structure- based drug design and chemical synthesis to generate a library of optimized C-B06 analogues for evaluation in a FRET-suppression IRE-1 RNase assay, and carry out counterscreening with potent leads. Compounds will then be tested for their ability to block XBP-1s expression in intact cells. In Aim 2, we will determine the cytotoxicity of potent and selective covalent IRE-1 RNase inhibitors with purified E-TCL1 CLL cells, CLL cell lines, and freshly isolated primary patient samples collected at our Cancer Center. We will establish induction of apoptosis, XBP-1-dependant cytotoxicity, and the effect of our inhibitors on transcriptional targets downstream of XBP-1s. We will also carry out the above studies with existing allosteric antagonists to investigate whether reversible covalent inhibition is critical for the efficacy of IE-1 RNase inhibitors.